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. 2016 Apr 22;291(17):8862-75.
doi: 10.1074/jbc.M115.681726. Epub 2016 Jan 29.

An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory

Affiliations

An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory

Adrian J Butcher et al. J Biol Chem. .

Abstract

Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning.

Keywords: G protein-coupled receptor (GPCR); drug discovery; hippocampus; learning; mass spectrometry (MS); memory; phosphorylation.

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Figures

FIGURE 1.
FIGURE 1.
Agonist-mediated phosphorylation of the M1 mAChR. A, CHO cells expressing a C-terminally tagged mouse M1 mAChR (CHO-M1 cells) were treated with various concentrations of the muscarinic receptor agonists, acetylcholine (ACh) and xanomeline (Xan). Cells were then lysed and the inositol phosphate levels determined. Shown is the mean data of three experiments ± S.E. B, phosphorylation of the M1 mAChR was monitored in CHO-M1 cells metabolically labeled with [32P]orthophosphate and treated with vehicle, acetylcholine (100 μm), or xanomeline (Xan, 10 μm) followed by immunoprecipitation, gel electrophoresis, and autoradiography. Also shown are the mean data of five experiments ± S.E. The total M1 mAChR was also established using an anti-HA in a Western blot (Loading Control). C, summary of the mass spectrometric determination of the phosphorylation sites on the M1 mAChR. The phospho-sites indicated in red were the sites to which phospho-specific antibodies were raised. D, representative MS/MS spectrum and fragmentation table of M1 mAChR peptide phosphorylated on Ser228. E, summary of all the M1 mAChR phosphopeptides obtained from five experiments. Phosphorylated amino acids are highlighted in red. F, primary amino acid sequence of the human M1 mAChR receptor highlighting the phosphoacceptor sites in red.
FIGURE 2.
FIGURE 2.
Characterization of M1 mAChR phosphorylation-specific antibodies. A, schematic representation of the experimental procedure used to characterize M1 mAChR phosphorylation-specific antibodies where CIAP was used to dephosphorylate immunoprecipitated M1 mAChR receptor derived from cells that had been treated with acetylcholine (ACh) before being probed in Western blots with the phospho-specific antibodies. B, results of Western blots from the experiments that are illustrated in A. Blots were probed with phospho-specific antibodies directed toward Ser(P)228, Ser(P)273, Ser(P)322, and Ser(P)451. Also shown is the loading control from this particular experiment using an anti-HA antibody to detect the epitope-tagged M1 mAChR. The * indicates slight cross-reactivity of the phospho-specific antibodies with CIAP.
FIGURE 3.
FIGURE 3.
Agonist-dependent phosphorylation revealed by phospho-specific antibodies to the M1 mAChR. M1 mAChRs were immunoprecipitated from non-transfected (NT) CHO cells or CHO-M1 cells that were treated with vehicle, acetylcholine (ACh, 100 μm), or xanomeline (Xan, 10 μm) for 5 min, resolved by SDS-PAGE, and probed in Western blots with phospho-specific antibodies raised against Ser(P)228, Ser(P)273, Ser(P)322, and Ser(P)451. Total M1 mAChR was determined in the loading control using anti-HA antibodies. The experiment shown is typical of at least five independent experiments.
FIGURE 4.
FIGURE 4.
Positive allosteric modulator (BQCA) promotes M1 mAChR phosphorylation at Ser228. A, schematic representation of the M1 mAChR illustrating distinct binding modes for orthosteric ligands, acetylcholine (ACh) and xanomeline (Xan), from that of allosteric ligands, such as BQCA. The chemical structure of BQCA is also shown. B, CHO-M1 cell membranes were used in competition radioligand binding experiments where acetylcholine displacement of [3H]NMS was tested in the presence of a range of concentrations of BQCA. The data presented are means ± S.E. of three independent experiments conducted in triplicate. C, PAM activity of BQCA was revealed in concentration-response curves for acetylcholine-mediated inositol phosphate production in CHO-M1 cells in the presence of various concentrations of BQCA. D, both the mixed agonist (green arrow) and PAM (black arrow) activity of BQCA was revealed in concentration-response curves for acetylcholine pERK1/2 response in CHO-M1 cells in the presence of various concentrations of BQCA. E, representative Western blots of CHO-M1 cell lysates stimulated with increasing concentrations of acetylcholine in the presence or absence of BQCA (3 μm) and probed with the phospho-specific serine 228 antibody or anti-HA antibodies as a loading control for total M1 mAChR. Also shown are the mean data of three independent experiments ± S.E. NT, non-transfected cells. F, CHO-M1 cells were stimulated with a high concentration of acetylcholine (500 μm) or with BQCA at two concentrations; a low concentration (3 μm) where BQCA might be expected to show no intrinsic agonist activity and a high concentration (100 μm) where BQCA would show agonist activity. Cell lysates were prepared, resolved by SDS-PAGE, and Western blots probed with the phospho-specific serine 228 antibody. The IgG band was used as a loading control in these experiments. Also shown is the mean data ± S.E. of Western blots from three independent experiments. G, CHO-M1 cells stimulated with acetylcholine or BQCA were fixed and processed for immunocytochemistry using an anti-HA antibody to show total M1 mAChR or with the phospho-specific serine 228. Non-transfected CHO cells were similarly treated. All the images and gels shown were typical of at least three independent experiments. All the graphical data represent the mean ± S.E. of at least three independent experiments. Statistical analysis uses Student's paired t test.
FIGURE 5.
FIGURE 5.
Phosphorylation of Ser228 on the M1 DREADD receptor could be detected in the hippocampus following receptor activation with a selective agonist. A, illustration of the two point mutations (Y106C and A195G) used to generation the HA epitope-tagged M1 DREADD receptor mutant in which activation by acetylcholine (ACh) is abolished but instead the receptor could be activated by CNO. B, CHO FlpIn cells expressing the HA-tagged M1 DREADD receptor were stimulated with increasing concentrations of acetylcholine or CNO. Western blots (WB) were probed with either anti-HA (as a loading control) or phospho-specific serine 228 antibodies. NT, non-transfected cells. C and D, M1 DREADD knock-in mice or M1 mAChR-knock-out mice (M1-KO) were injected (intraperitoneally) with CNO (0.3 mg/kg). After 30 min tissue was fixed by transcardial perfusion and sections stained with anti-HA antibodies (C) or phospho-specific serine 228 antibodies (D). E, fixed sections from M1 mAChR knock-out mice (M1-KO) or M1 DREADD knock-in mice treated with vehicle or CNO (0.3 mg/kg) were co-stained with anti-HA (green) and anti-phospho-specific serine 228 (red) antibodies. Two neurons where the staining for the receptor and the phosphorylated receptor occur in the same neuron are indicated by the arrows. The areas marked by the white box are magnified in the lower panels.
FIGURE 6.
FIGURE 6.
M1 mAChR is activated in the hippocampus following drug treatment and memory acquisition. A, C57/BL6/NTAC mice were injected (i.p.) with vehicle or xanomeline (5 mg/kg). After 30 min, tissues were fixed by transcardial perfusion, and sections were obtained and stained with the phospho-specific serine 228 antibody and with DAPI stain to reveal the nuclei. Shown are representative sections through the CA1 region of the hippocampus. B, C57/BL6/NTAC mice (Wild-Type) or M1 mAChR-knock-out mice (M1-KO) were injected (intraperitoneally) with BQCA (15 mg/kg) or vehicle, and after 30 min hippocampal membranes were prepared from which the M1 mAChR was immunoprecipitated. The sample was then processed in Western blots, which were probed with phospho-specific serine 228 antibody or an M1 mAChR-specific antibody to detect total M1 mAChR. C, quantification of Western blots from B. The data are presented as means ± S.E. (n = 3). Statistical analysis uses Student's paired t test. D, C57/BL6/NTAC mice were subjected to a fear conditioning training protocol or to an unpaired immediate foot shock as a control; 30 min later tissue was fixed by transcardial perfusion, and sections obtained and stained with phosphorylated Ser228-specific antibody (upper panel) or anti-c-FOS antibody (lower panel). All the data shown are typical of at least three independent experiments.

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References

    1. Jacoby E., Bouhelal R., Gerspacher M., and Seuwen K. (2006) The 7 TM G-protein-coupled receptor target family. ChemMedChem 1, 761–782 - PubMed
    1. Rasmussen S. G., DeVree B. T., Zou Y., Kruse A. C., Chung K. Y., Kobilka T. S., Thian F. S., Chae P. S., Pardon E., Calinski D., Mathiesen J. M., Shah S. T., Lyons J. A., Caffrey M., Gellman S. H., et al. (2011) Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 - PMC - PubMed
    1. Venkatakrishnan A. J., Deupi X., Lebon G., Tate C. G., Schertler G. F., and Babu M. M. (2013) Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 - PubMed
    1. Kenakin T. (2005) New concepts in drug discovery: collateral efficacy and permissive antagonism. Nat. Rev. Drug Discov. 4, 919–927 - PubMed
    1. Kenakin T., and Williams M. (2014) Defining and characterizing drug/compound function. Biochem. Pharmacol. 87, 40–63 - PubMed

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